Use of trans-activation and CIS-activation to modulate the persistence of expression of a transgene

ABSTRACT

Provided are methods of modulating the persistence of the expression in a cell of a transgene, such as a transgene in a non-Herpes vector or in at least E4Δ adenoviral vector, and related systems. One method comprises contacting the cell with a non-Herpes vector comprising and expressing a gene encoding HSV ICP0, whereupon expression of HSV ICP0 the persistence of expression of the transgene is modulated. Further provided is a system for modulating the persistence of expression of a transgene, which system comprises a non-Herpes vector comprising (i) a gene encoding HSV ICP0 and (ii) a transgene, wherein the HSV ICP0 modulates the persistence of expression of the transgene and either the non-Herpes vector comprises the transgene or the system further comprises a vector, in which case the vector comprises the transgene.

This application is a continuation of International Patent ApplicationNo. PCT/US99/28637, filed Dec. 3, 1999, which is a continuation-in-partof U.S. patent application Ser. No. 09/205,014, filed Dec. 4, 1998, nowU.S. Pat. No. 6,225,113.

TECHNICAL FIELD OF THE INVENTION

This invention relates to cis- and trans-activation methods ofmodulating the persistence of expression of a transgene in non-Herpesvectors, more particularly in at least E4-deficient (E4Δ) adenoviralvectors, as well as transactivation systems for use in such methods.

BACKGROUND OF THE INVENTION

Gene therapy relies upon the introduction of one or more recombinantnucleic acid molecules (i.e., vectors) comprising one or more transgenesinto a host (e.g., a human), wherein the presence, and in most cases theexpression, of the transgene produces some desired effect (e.g.,production of a therapeutic protein). Thus, for gene therapy to beeffective, vectors (particularly, gene transfer vectors) that providepersistent expression of the transgene are desired.

Using vector reporter gene constructs, it has been established that highlevels of vector transgene expression can be obtained in a variety ofanimal models. However, it also has been established that the high levelof transgene expression so obtained is transient, with reporter geneexpression peaking within the first week after infection and becomingessentially undetectable about 80 days after infection. Recent studieshave indicated that the limited persistence of gene expression in vivois most likely due to an immune response of the host against virallyinfected cells. For example, gene expression can be maintained inimmunologically privileged neuronal or retinal tissues for periods inexcess of two months and in immunodeficient or immunologically naïverodents for periods in excess of six months. Transgene expression inimmune-competent animals, by contrast, rapidly declines to baselinelevels within 2-3 weeks of infection due to immune activation.

Using a combination of mouse strains, which are defective in specificelements of the immune system, it has been shown that the immuneresponse against cells infected with viral vectors involves bothcellular and humoral components of the immune system. For example,immunodeficient mice, which lack mature T- and B-lymphocytes, expressadenovirus-mediated transgenes beyond four months (Kass-Eisler et al.,Gene Therapy 1: 395-402 (1994); Yang et al., Immunity 1: 433-442(1994a); Yang et al., PNAS USA 91: 4407-4411 (1994b); Dai et al., PNASUSA 92: 1401-1405 (1995); Kay et al., Nat. Genet. 11: 191-197 (1995);and Yang et al., J. Immunol. 155: 2564-2570 (1995)). Similarly, transferof CD8⁺ and CD4⁺ cytotoxic T-cells from adenoviral vector-infected miceto infected RAG-2 mice, which lack mature B- and T-lymphocytes, resultsin clearance of the vector and the transgene by apoptosis (Yang et al.(1994a), supra; and Yang et al. (1995), supra), whereas immune depletionof CD8⁺ or CD4⁺ cells in immunocompetent mice results in persistenttransgene expression (Yang et al. (1994a), supra; Kay et al., Nat.Genet. 11: 191-197 (1995); Yang et al. (1995), supra; Kolls et al., Hum.Gene Ther. 7: 489-497 (1996); and Guerette et al., Transplantation 62:962-967 (1996)). While pathways involving perforin and Fas are the majorpathways responsible for T-cell cytotoxicity (Kojima et al., Immunity 1:357-364 (1994); Henkart, Immunity 1: 343-346 (1994); Kagi et al.,Science 265: 528-530 (1994); and Kagi et al., Eur. J. Immunol. 25:3256-3262 (1995)), the perforin/granzyme pathway has been reported tomediate clearance of adenoviral gene transfer vectors byantigen-specific, cytotoxic T-cells (Yang et al., PNAS USA 92: 7257-7261(1995)).

In addition to limiting the persistence of gene expression from viralvectors, the immune response inhibits successful readministration ofviral vectors, which limits the period of gene expression. For example,adenoviruses are classified into 47 different serotypes and a number ofsubgroups, namely A through G, based on a number of criteria, includingantigenic cross-reactivity. Following an initial administration ofadenovirus, serotype-specific antibodies are generated against epitopesof the major viral capsid proteins, namely the penton, hexon and fiber.Given that such capsid proteins are the means by which the adenovirusattaches itself to a cell and subsequently infects the cell, suchantibodies (i.e., neutralizing antibodies) are then able to block or“neutralize” reinfection of a cell by the same serotype of adenovirus.This necessitates using a different serotype of adenovirus in order toadminister one or more subsequent doses of exogenous DNA to continue toexpress a given gene, such as in the context of gene therapy.

Another approach to increasing the persistence of transgene expressionin vector systems involves the introduction of substantial deletions ina viral vector so as to reduce or eliminate completely the production ofviral antigens by the viral vector. In this regard, the deletion of E4from adenoviral vectors is especially important for safe adenoviralvector design. Removal of the E4 region severely disrupts adenoviralgene expression in transduced cells. Removal of the E4 region alsoeliminates several viral products that interact with and antagonizecellular targets and processes. E4-ORF6 has been shown to block p53function and to have oncogenic potential (Dobner et al., Science 272:1470-1473 (1996); Nevels et al., PNAS USA 94: 1206-1211 (1997)). It alsoappears that E4-ORF1 has oncogenic potential (Javier et al., J. Virol.65: 3192-3202 (1991); Javier et al., Science 257: 1267-1271 (1992);Javier et al., Breast Cancer Res. Treat. 39: 57-67 (1996); Javier etal., J. Virol. 68: 3917-3924 (1994); Weiss et al., J. Virol. 71:4385-4393 (1997); Weiss et al., J. Virol. 71: 1857-1870 (1997); andWeiss et al., J. Virol. 70: 862-872 (1996)). ORF6 and ORF3 of the E4region of adenovirus also have been shown to be involved in alteringmRNA expression post-transcriptionally (Nordqvist et al., PNAS USA 87:9543-9547 (1990); Nordqvist et al., Mol. Cell. Biol. 14: 437-445 (1994);Nordqvist et al., Mol. Biol. Rep. 14: 203-204 (1990); Ohman et al.,Virology 194: 50-58 (1993); Sandler et al., J. Virol. 63: 624-630(1989); and Sandler et al., Virology 181: 319-326 (1991)). E4 productsare also involved in controlling E2F (Nevins, Virus Res. 20: 1-10(1991)), E1A-induced p53-independent apoptosis (Marcellus et al., J.Virol. 70: 6207-6215 (1996)), the modulation of the phosphorylationstatus of cellular and viral proteins (Kleinberger et al. 67: 7556-7560(1993); and Muller et al., J. Virol. 66: 5867-5878 (1992)), and thealteration of the nuclear transport of various proteins (Goodrum et al.,J. Virol. 70: 6323-6335 (1996)). Elimination of the E4 region ofadenovirus eliminates these negative effects. However, E4 eliminationalso adversely affects maintenance of transgene persistence.

Provision of E4 in trans has been proposed as a method of activatingtransgene expression from an E4Δ adenoviral vector (Brough et al., J.Virol. 71(12): 9206-9213 (1997)). Supply of E4 products in trans hasbeen demonstrated to allow persistent expression from thecytomegalovirus E4 promoter (Armentano et al., J. Virol. 71(3):2408-2416 (1997)). One potential problem associated with anyadministration of E4 products is that a multiplicity of E4 containingvectors has been found to result in cell toxicity, particularly inendothelial cells. Co-expression of the adenoviral E2 preterminalprotein from an adenoviral vector or in trans has been demonstrated tostabilize in vitro an adenoviral mini-genome, which is deficient in E1,E2 and E3 but not E4 (Lieber et al., Nature Biotech. 15: 1383-1387(1997)). Expression of a transgene operably linked to thecytomegalovirus (CMV) immediate early promoter has been demonstrated tobe dependent on the infected cell protein 0 in Herpes simplex vectors;based on such a showing, it was suggested that ORF3 of the E4 region ofadenovirus could have the same effect on transgene expression in anadenoviral vector (Samaniego et al., J. Virol. 72(4): 3307-3320 (1998)).

Adenoviruses in many settings are advantageous as viral vectors becausethey are easy to use, can be produced in high titers (i.e., up to about10¹³ viral particles/ml), transfer genes efficiently to nonreplicating,as well as replicating, cells (see, for example, review by Crystal,Science 270: 404-410 (1995)), and exhibit a broad range of host- andcell-type specificity. Such advantages have resulted in a recombinantadenovirus being the vector of choice for a variety of gene transferapplications. Adenoviral vectors are especially preferred for somaticgene therapy of the lungs, given their normal tropism for therespiratory epithelium.

Other advantages that accompany the use of adenoviruses as vectors in invivo gene expression include: (1) the rare observance of recombination;(2) the absence of an ostensible correlation of any human malignancywith adenoviral infection, despite the common occurrence of infection;(3) the adenoviral genome (which is comprised of linear, double-strandedDNA) can be manipulated to carry up to about 7.5 kb of exogenous DNA,and longer DNA sequences can potentially be carried into a cell, forinstance, by attachment to the adenoviral capsid (Curiel et al., HumanGene Therapy 3: 147-154 (1992)); (4) an adenovirus can be modified suchthat it does not interfere with normal cellular function, given that thevector controls expression of its encoded sequences in an epichromosomalmanner; and (5) it already has been proven safe to use in humans, giventhat live adenovirus has been safely used as a human vaccine for manyyears.

Intravenous administration of adenovirus to mice results in the vastmajority of adenovirus being localized to the liver (Worgall et al.,Human Gene Therapy 8: 37-44 (1997)). During the first 24-48 hrs ofinfection, 90% of vector DNA is eliminated, presumably through innatepathways of viral clearance mediated by Kupffer cells in the liver(Worgall et al. (1997), supra), well before maximal levels of transgeneare expressed. In spite of the fact that the majority of virus iscleared within one to two days, over 95% of hepatocytes are transducedby the remaining small percentage of input adenoviral vectors (Li etal., Human Gene Therapy 4: 403-409 (1993)) with maximum transgeneexpression occurring during the first week of post-infection.

In view of the above, there is a need in the art for other methods andcompositions than those known in the art that effect persistenttransgene expression in vector systems. More particularly, there is aneed for such methods and compositions for vectors that are useful ingene therapy, such as adenoviral gene transfer vectors.

The present invention seeks to address some of the disadvantagesinherent to the methods and vectors of the prior art by providing, amongother things, methods and vectors or systems that modulate thepersistence of expression of a transgene in a cell. This and otherobjects and advantages of the present invention, as well as additionalinventive features, will be apparent from the following detaileddescription.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of modulating the persistence ofthe expression in a cell of a transgene, such as a transgene in anon-Herpes vector or in at least E4Δ adenoviral vector. One methodcomprises contacting the cell with a non-Herpes vector comprising andexpressing a gene encoding HSV ICP0, whereupon expression of HSV ICP0the persistence of expression of the transgene is modulated. In thisregard, the present invention further provides a system for modulatingthe persistence of expression of a transgene, which system comprises anon-Herpes vector comprising (i) a gene encoding HSV ICP0 and (ii) atransgene, wherein the HSV ICP0 modulates the persistence of expressionof the transgene and either the non-Herpes vector comprises thetransgene or the system further comprises a vector, in which case thevector comprises the transgene.

Another method comprises contacting the cell with an at least E4Δadenoviral vector comprising (i) a transgene and (ii) a gene encoding atrans-acting factor, which is not from the E4 region of an adenovirusand which modulates the persistence of expression of the transgene. Inanother embodiment of this method, i.e., a two vector embodiment, themethod comprises contacting the cell simultaneously or sequentially with(i) an at least E4Δ adenoviral vector comprising a transgene and (ii) aviral vector comprising a gene encoding a trans-acting factor, which isnot from the E4 region of an adenovirus and which modulates thepersistence of expression of the transgene. In this regard, the presentinvention further provides a system for modulating the persistence ofexpression of a transgene in an at least E4Δ adenoviral vector, whichsystem comprises (i) an at least E4Δ adenoviral vector comprising atransgene and (ii) a viral vector comprising a gene encoding atrans-acting factor. The gene encoding the trans-acting factor is notfrom the E4 region of an adenovirus and either the at least E4Δadenoviral vector comprises the gene encoding the trans-acting factor orthe system further comprises a viral vector, in which case the viralvector comprises the gene encoding the trans-acting factor. Thetrans-acting factor modulates the persistence of expression of thetransgene.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, at least in part, on theobservation that an at least E4Δ adenoviral vector expresses a transgeneat high levels for a limited amount of time in vivo. The presentinvention is further predicated on the discovery that persistence ofexpression of a transgene in an at least E4Δ adenoviral vector can bemodulated through the action of a trans-acting factor, such as HSV ICP0or Ad pTP, among others. The present invention is additionally basedupon the discovery that persistence of expression of a transgene in acell can be modulated by contacting the cell with any suitablenon-Herpes vector, including an adenoviral gene transfer vector, thatcomprises and expresses a gene encoding HSV ICP0.

Accordingly, the present invention provides a method of modulating thepersistence of expression of a transgene in a cell. By “transgene” ismeant any gene that can be expressed in a cell. Desirably, theexpression of the transgene is beneficial, e.g., prophylactically ortherapeutically beneficial, to the cell or a tissue, organ, organsystem, organism or cell culture of which the cell is a part. If thetransgene confers a prophylactic or therapeutic benefit to the cell, thetransgene can exert its effect at the level of RNA or protein. Forexample, the transgene can encode a protein that can be employed in thetreatment of an inherited disease, e.g., the cystic fibrosistransmembrane conductance regulator can be employed in the treatment ofcystic fibrosis. Alternatively, the transgene can encode an antisensemolecule, a ribozyme, a protein that affects splicing or 3′ processing(e.g., polyadenylation), or a protein that affects the level ofexpression of another gene within the cell (i.e., where gene expressionis broadly considered to include all steps from initiation oftranscription through production of a process protein), such as bymediating an altered rate of mRNA accumulation or transport or analteration in post-transcriptional regulation. The transgene can be partof an expression cassette. In the context of the present invention, ifthe transgene is located in the non-Herpes vector, the transgene can belocated anywhere in the non-Herpes vector. Preferably, in thoseembodiments of the present invention when the non-Herpes vector is anadenoviral vector (such as an adenoviral gene transfer vector), thetransgene is located in the E1 region of the adenoviral vector.

Any suitable non-Herpes vector can be utilized in the context of thepresent invention. A “non-Herpes” vector is any vector that is notsubstantially derived from Herpes simplex virus (HSV) and/or does notcontain a substantial proportion of the Herpes simplex genome.Preferably, the non-Herpes vector of the present invention comprisesthree genes or less derived from HSV, more preferably two or less, andoptimally only HSV ICP0 or a portion thereof responsible fortrans-activation in the context of the present invention. Suitableexamples of non-Herpes vectors include plasmids, cosmids, and viralvectors. Desirably, the non-Herpes vector is a viral vector, morepreferably a DNA viral vector, and even more preferably a DNA viralvector that does not integrate into the host genome. More preferablystill, the non Herpes vector is an adenoviral vector, more particularlyan adenoviral gene transfer vector, such as an at least E4Δ adenoviralvector.

In one aspect, the present invention provides a method of modulating thepersistence of expression of a transgene in a cell by contacting thecell with a non-Herpes vector comprising and expressing a gene encodingHSV ICP0, whereupon expression of HSV ICP0 the persistence of expressionof the transgene is modulated. HSV ICP0 indicates the infected cellpolypeptide of Herpes simplex virus, and thus the present inventionincludes use of the HSV ICP0 gene or an RNA or cDNA corresponding to theHSV ICP0 gene. The method can further comprise contacting the cell witha vector comprising a transgene. The non-Herpes vector can furthercomprise a cis-acting factor as described herein. Ad pTP can be used ina similar manner as HSV ICP0.

HSV ICP0 is comprised of multiple domains, encoded by different portionsof the HSV ICP0 gene. It is believed that one or more of these domainsmay be sufficient to induce the effect(s) that are responsible for themodulation of transgenes in non-Herpes vectors. The identification ofsuch domains can be accomplished using standard techniques known in theart. For example, by performing mutational analysis on the HSV ICP0 geneencoding the different domains (or performing similar analysis on thegene products of such domains), domains and/or regions within thedomains that are responsible for such effects can be identified. Anexample of similar analysis using such techniques is described in Vos etal., Virology, 172(2): 634-42 (1989). By performing such techniquesdomain(s) of HSV ICP0 can be identified that can be used as trans-actingfactors in modulating the persistence of transgene expression. In asimilar vein, genes that demonstrate a high level of sequence homologyto HSV ICP0 can be later identified from other organisms, orsynthetically produced, and used as trans-acting factors in modulatingthe persistence of transgene expression. The identification of suchgenes can lead to the identification of cellular functions involved intransgene persistence. The presence of HSV ICP0 in non-Herpes vectorsleads to lower production of neutralizing antibodies by a host against anon-Herpes vector, such as an adenoviral vector. The present invention,thus, has applicability in the administration of immunogenic naked DNAs.

The present invention further provides a system for modulating thepersistence of expression of a transgene. The system comprises anon-Herpes vector comprising (i) a gene encoding HSV ICP0 and (ii) atransgene, wherein, the HSV ICP0 modulates the persistence of expressionof the transgene and either the non-Herpes vector comprises thetransgene or the system further comprises a vector, in which case thevector comprises the transgene. Such a system also can further comprisea cis-acting factor, as described elsewhere herein, either present onthe non-Herpes vector and/or on the vector. Such a system can include acarrier for the system, preferably a pharmaceutically acceptablecarrier, examples of which are widely known in the art and areexemplified herein.

In another aspect, the present invention provides a method formodulating the persistence and expression of a transgene in at least E4Δadenoviral vector in a cell. By “an least E4Δ adenoviral vector” ismeant an adenoviral vector that is at least deficient in the E4 regionof the adenoviral genome. The vector can also be deficient in one ormore other regions of the adenoviral genome, such as early regionsand/or late regions. By “deficient” is meant an absence of a genefunction in a given region of the adenoviral genome. In other words, theregion does not comprise, encode and/or express a wild-type adenoviralgene function. In the case of the E4 region, this includes functionsrequired for viral DNA replication, mRNA splicing and accumulation, lateprotein expression and inhibition of host cell protein synthesis. In theE4 region, the deficiency is desirably complete. Any deficiency in oneor more other regions of the adenoviral genome can be complete orpartial. The absence of gene function can be due to a deletion, aninsertion or a mutation, for example. The one or more deficiencies inthe adenoviral vector should be such that a transgene or an expressioncassette comprising a transgene can be inserted into the adenoviralvector and expressed.

In one embodiment of the method, i.e., a single vector embodiment, themethod comprises contacting the cell with an at least E4Δ adenoviralvector comprising (i) a transgene and (ii) a gene encoding atrans-acting factor. In another embodiment, i.e., a two vectorembodiment, the method comprises contacting the cell simultaneously orsequentially with (i) an at least E4Δ adenoviral vector comprising atransgene and (ii) a viral vector comprising a gene encoding atrans-acting factor. In both embodiments of the method, the trans-actingfactor modulates the persistence of expression of the transgene and thegene encoding the trans-activating factor is not from the E4 region ofan adenovirus.

“Contacting” can be done by any means known to those skilled in the art,and described herein, by which the apparent touching or mutual tangencyof the vector(s) with the cell can be effected. Optionally, the vectorcan be further complexed with a bi-specific or multi-specific molecule(e.g., an antibody or fragment thereof), in which case “contacting”involves the apparent touching or mutual tangency of the complex of thevector and the bi-specific or multi-specific molecule with the cell. Forexample, the vector and the bi-specific (multi-specific) molecule can becovalently joined, e.g., by chemical means known to those skilled in theart, or other means. Preferably, the vector and the bi-specific(multi-specific) molecule can be linked by means of noncovalentinteractions (e.g., ionic bonds, hydrogen bonds, Van der Waals forces,and/or nonpolar interactions). Although the vector and the bi-specific(multi-specific) molecule can be brought into contact by mixing in asmall volume of the same solution, the cell and the complex need notnecessarily be brought into contact in a small volume, as, for instance,in cases where the complex is administered to a host (e.g., a human),and the complex travels by the bloodstream to the cell to which it bindsselectively and into which it enters. The contacting of the vector witha bi-specific (multi-specific) molecule preferably is done before thecell is contacted with the complex of the adenovirus and the bi-specific(multi-specific) molecule.

With respect to the two vector embodiment, “simultaneously” means thatthe at least E4Δ adenoviral vector and the viral vector are brought intocontact with a cell at the same time (or sufficiently close in time asto be considered at the same time). “Sequentially” means that the atleast E4Δ adenoviral vector and the viral vector are brought intocontact with a cell one after the other. If the two vectors aresequentially administered, preferably the viral vector is administeredsubsequently to the at least E4Δ adenoviral vector comprising thetransgene. Sequential administration of the second vector, such as theviral vector, can be immediate or delayed and by the same route or adifferent route, e.g., intravenous or intramuscular. If sequentialadministration of the second vector is delayed, the delay can be amatter of minutes, hours, days, weeks, months or even longer. In thisregard, sequential administration of the second vector can be delayedthrough the use of a time-release composition. The two vector embodimentof the method, therefore, allows for modulation in situations wherethere is substantial delay between the administration of the at leastE4Δ adenoviral vector comprising the transgene and the viral vectorcomprising the gene encoding the trans-acting factor such thatexpression of the transgene has substantially decreased over time.

A cell can be any cell and can be present as a single entity, or can bepart of a larger collection of cells. Such a larger collection of cellscan comprise, for instance, a cell culture (either mixed or pure), atissue (e.g., epithelial or other tissue), an organ (e.g., heart, lung,liver, gallbladder, urinary bladder, eye or other organ), an organsystem (e.g., circulatory system, respiratory system, gastrointestinalsystem, urinary system, nervous system, integumentary system or otherorgan system), or an organism (e.g., a bird, mammal, particularly ahuman, or the like). Preferably, the organs/tissues/cells are of thecirculatory system (e.g., including, but not limited to heart, bloodvessels, and blood), respiratory system (e.g., nose, pharynx, larynx,trachea, bronchi, bronchioles, lungs, and the like), gastrointestinalsystem (e.g., including mouth, pharynx, esophagus, stomach, intestines,salivary glands, pancreas, liver, gallbladder, and others), urinarysystem (e.g., such as kidneys, ureters, urinary bladder, urethra, andthe like), nervous system (e.g., including, but not limited to, brainand spinal cord, and special sense organs, such as the eye) andintegumentary system (e.g., skin). Even more preferably, the cells areselected from the group consisting of heart, blood vessel, lung, liver,gallbladder, urinary bladder, and eye cells.

If a vector in accordance with the present invention is targeted to acell (e.g., in a manner described above with respect to “contacting”),the cell to which the vector is targeted differs from another cell,which is not targeted, in that the cell so being targeted comprises aparticular cell-surface binding site (e.g., that is recognized by thebi-specific (multi-specific) molecule). By “particular cell-surfacebinding site” is meant any site (i.e., molecule or combination ofmolecules) present on the surface of a cell with which the vector, e.g.,adenoviral vector, can interact in order to attach to the cell and,thereby, enter the cell. A particular cell-surface binding site,therefore, encompasses a cell-surface receptor and, preferably, is aprotein (including a modified protein), a carbohydrate, a glycoprotein,a proteoglycan, a lipid, a mucin molecule or mucoprotein, or the like.Examples of potential cell-surface binding sites include, but are notlimited to: heparin and chondroitin sulfate moieties found onglycosaminoglycans; sialic acid moieties found on mucins, glycoproteins,and gangliosides; major histocompatability complex I (MHC I)glycoproteins; common carbohydrate molecules found in membraneglycoproteins, including mannose, N-acetyl-galactosamine,N-acetyl-glucosamine, fucose, and galactose; glycoproteins, such asICAM-1, VCAM, E-selectin, P-selectin, L-selectin, and integrinmolecules; and tumor-specific antigens present on cancerous cells, suchas, for instance, MUC-1 tumor-specific epitopes. However, targeting anadenovirus to a cell is not limited to any specific mechanism ofcellular interaction (i.e., interaction with a given cell-surfacebinding site).

Trans-acting factors are known in the art. Some trans-acting factors aresecreted by cells that express them; others are not. The trans-actingfactor modulates the persistence of expression of the transgene. In thecontext of the present invention, it is desired that the trans-actingfactor not be secreted by the cell that expresses it, in which case, thetrans-acting factor (and any cis-acting factor) must be expressed in thesame cell as the transgene. The gene encoding the trans-acting factor isnot from the E4 region of an adenovirus. A preferred trans-acting factorthat is not from the E4 region of an adenovirus is Ad pTP. Preferably,the gene encoding the trans-acting factor is not from an adenovirus. Thetrans-acting factor can be of viral or cellular origin and can be underthe control of a regulatable promoter, such as an inducible promoter(e.g., tet) or a repressible promoter, a regulatable expression system(e.g., tetracycline, rampamycin or radiation-inducible), or a cell- ortissue-type expression system as are known in the art (Rossi et al.,Current Opinion in Biotechnology 9:451-456 (1998)). Examples oftrans-acting factors that are not from an adenovirus include HSV ICP0(Jordan et al., J. Virol. 71:6850-6862 (1997); and Moriuchi et al.,Virology 209: 281-283 (1995)), Cytomegalovirus unique sequence longdomain 84 (CMV-UL84) (Sarisky et al., J. Virol. 70(11): 7393-7413(1996); and Schmolke et al., J. Virol. 71(9): 7048-7060 (1997)),Varicella-Zoster virus ORF 61 (VZV-ORF61) (Moriuchi et al. (1995),supra), Pseudorabies virus early protein 0 (PRV-EP0) (Moriuchi et al.(1995), supra), Human Cytomegalovirus immediate early protein (CMV-IE) 1(Ahn et al, Mol. Cell. Biol. 18(8): 4899-4913 (1998)), CMV-IE2, CMV-IE86(Bresnahan et al, J. Biol. Chem. 272(34): 22075-22082 (1998)), HIV-tat(Schafer et al., J. Virol. 70(10): 6937-6946 (1996)), HTLV-tax, HBV-X,AAV-Rep 78 (Weger et al., J. Virol. 71(11): 8437-8447 (1997)); Pereiraet al., J. Virol. 71(2): 1079-1088 (1997); and Pasquale et al., J.Virol. 72(10): 7916-7925 (1998)), the cellular factor from the U2OSosteosarcoma cell line (U2OS) that functions like HSV ICP0 (Yao et al.,J. Virol. 69(10): 6249-6258 (1995)), and the cellular factor in PC12cells that is induced by nerve growth factor (Jordan et., J. Virol.72(7): 5373-5382 (1998)). The HSV ICP0-like factor from the U2OSosteosarcoma cell line and the cellular factor in PC12 cells that isinduced by nerve growth factor can be isolated, for example, by makingcDNA from the cell line, cloning the cDNA into an adenoviral cosmid,constructing an adenoviral vector library that expresses the cDNA, andscreening for the factor, such as by complementing for growth of anICP0-deleted herpes vector and/or maintaining expression of CMV-drivenGFP from the ICP0-deleted herpes vector, pulling out the complementedcells, and recovering the adenovirus vector containing and expressingthe factor. Whether or not a given trans-acting factor can modulate agiven transgene, including a transgene that is part of an expressioncassette, can be determined in accordance with methods set forth in theExamples and other methods known in the art. A preferred trans-actingfactor that is not from an adenovirus is HSV ICP0. Preferably, thetransgene comprises a promoter from a cytomegalovirus or a Rous sarcomavirus or the transgene is part of an expression cassette that comprisessuch a promoter. The gene encoding the trans-acting factor preferablycomprises an adenoviral E4 promoter. Preferably, the at least E4Δadenoviral vector further comprises a cis-acting factor. The cis-actingfactor can be of viral or cellular origin and can be under the controlof a regulatable promoter, such as an inducible promoter or arepressible promoter, examples of which are recited above. Examples ofcis-acting factors include a matrix attachment region (MAR) (e.g.,immunoglobulin heavy chain μ (murine; Jenuwein et al., Nature 385(16):269 (1997)), apolipoprotein B (human; Kalos et al., Molec. Cell. Biol.15(1): 198-207 (1995)), papillomavirus type 16 (human; Tan et al., J.Virol. 72(5): 3610-3622 (1998)), and clotting factor VIII (human;Fallaux et al., Molec. Cell. Biol. 16(8): 4264-4272 (1996)), a locuscontrol region (LCR), and a scaffold attachment region (SAR) (e.g.,β-interferon (human; Agarwal et al., J. Virol 42(5): 3720-3728 (1998))).In the two vector embodiment, the viral vector preferably is anadenoviral vector or a Herpes simplex vector. The viral vector canfurther comprise a transgene, in which case the viral vector can furthercomprise a cis-acting factor.

In view of the above, the present invention further provides arecombinant at least E4Δ adenoviral vector for use in the single vectorembodiment of the method. The vector comprises (i) a transgene and (ii)a gene encoding a trans-acting factor. The trans-acting factor modulatesthe persistence of expression of the transgene. The gene encoding thetrans-acting factor is not from the E4 region of an adenovirus. Apreferred trans-acting factor that is not from the E4 region of anadenovirus is pTP. Preferably, the gene encoding the trans-acting factoris not from an adenovirus. Examples of trans-acting factors that are notfrom an adenovirus include HSV ICP0, CMV UL84, VZV-ORF61, PRV-EP0,CMV-IE1, CMV-IE2, CMV-IE86, HIV-tat, HTLV-tax, HBV-X, AAV-Rep 78, thecellular factor from the U20S osteosarcoma cell line that functions likeHSV ICP0, and the cellular factor in PC12 cells that is induced by nervegrowth factor. A preferred trans-acting factor that is not from anadenovirus is HSV ICP0. The transgene preferably comprises a promoterfrom a cytomegalovirus or a Rous sarcoma virus or the transgene is partof an expression cassette that comprises such a promoter. The geneencoding the trans-acting factor preferably comprises an adenoviral E4promoter. Preferably, the vector further comprises a cis-acting factor.Examples of cis-acting factors include an MAR (e.g., immunoglobulinheavy chain μ (murine), apolipoprotein B (human), papillomavirus type 16(human) and clotting factor VIII (human)), an LCR or an SAR (e.g.,β-interferon (human)).

Also in view of the above, the present invention further provides asystem for modulation of a transgene in an at least E4Δ adenoviralvector. The system comprises (i) an at least E4Δ adenoviral vectorcomprising a transgene and (ii) a gene encoding a trans-acting factor.The trans-acting factor modulates the persistence of expression of thetransgene. The gene encoding the trans-acting factor is not the E4region of an adenovirus. The at least E4Δ adenoviral vector comprisesthe gene encoding the trans-acting factor or the system furthercomprises a viral vector, in which case the viral vector comprises thegene encoding the trans-acting factor. A preferred trans-acting factorthat is not from the E4 region of the adenovirus is pTP. Preferably, thegene encoding the trans-acting factor is not from an adenovirus.Examples of trans-acting factors that are not from an adenovirus includeHSV ICP0, CMV UL84, VZV-ORF61, PRV-EP0, CMV-IE1, CMV-IE2, CMV-IE86,HIV-tat, HTLV-tax, HBV-X, AAV-Rep 78, the cellular factor from the U20Sosteosarcoma cell line that functions like HSV ICP0, and the cellularfactor in PC12 cells that is induced by nerve growth factor. A preferredtrans-acting factor that is not from an adenovirus is HSV ICP0. Thetransgene preferably comprises a promoter from a cytomegalovirus or aRous sarcoma virus or the transgene is part of an expression cassettethat comprises such a promoter. The gene encoding the trans-actingfactor preferably comprises an adenoviral E4 promoter. Preferably, theviral vector is an adenoviral vector or a Herpes simplex vector. Theviral vector can further comprise a transgene and/or a cis-actingfactor. Preferably, the at least E4Δ adenoviral vector further comprisesa cis-acting factor. Examples of cis-acting factors include an MAR(e.g., immunoglobulin heavy chain μ (murine), apolipoprotein B (human),papillomavirus type 16 (human) and clotting factor VIII (human)), an LCRor an SAR (e.g., β-interferon (human)).

In the context of the present invention, the adenoviral vector can bederived from any adenovirus. An “adenovirus” is any virus of the familyAdenoviridae, and desirably is of the genus Mastadenovirus (e.g.,mammalian adenoviruses) or Aviadenovirus (e.g., avian adenoviruses). Theadenovirus can be of any serotype. Adenoviral stocks that can beemployed as a source of adenovirus can be amplified from the adenoviralserotypes 1 through 47, which are currently available from the AmericanType Culture Collection (ATCC, Rockville, Md.), or from any otherserotype of adenovirus available from any other source. For instance, anadenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31),subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, and 35), subgroupC (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9,10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroup E(serotype 4), subgroup F (serotypes 40 and 41), or any other adenoviralserotype. Preferably, however, an adenovirus is of serotype 2, 5 or 9.Desirably, an adenovirus comprises coat proteins (e.g., penton base,hexon, and/or fiber) of the same serotype. However, also preferably, oneor more coat proteins can be chimeric, in the sense, for example, thatall or a part of a given coat protein can be from another serotype.

Although the vector preferably is an adenoviral vector or a Herpessimplex viral vector, it can be any other suitable vector, preferably aviral vector. For example, the viral vector can be an adeno-associatedviral vector. In embodiments of the invention where the methods andcompositions are drawn to non-Herpes vectors comprising HSV ICP0, thevector is preferably an adenoviral vector.

Although the vector (e.g., the non-Herpes vector, and the at least E4Δadenoviral vector), in the context of the present invention, can bereplication-competent, preferably, these vectors arereplication-deficient or conditionally replication-deficient.Alternatively and preferably, the viral vector (and/or vector), which ispreferably an adenoviral vector or a Herpes simplex viral vector,comprises a genome with at least one modification therein, optimally amodification that renders the virus replication-deficient. Themodification to the viral genome includes, but is not limited to,deletion of a DNA segment, addition of a DNA segment, rearrangement of aDNA segment, replacement of a DNA segment, or introduction of a DNAlesion. A DNA segment can be as small as one nucleotide or as large as36 kilobase pairs, i.e., the approximate size of the adenoviral genome,or 38 kilobase pairs, which is the maximum amount that can be packagedinto an adenoviral virion. Preferred modifications, in addition to amodification that renders the vector replication-deficient, includeinsertion of (i) a transgene, (ii) a gene encoding a trans-acting factorand (iii) a cis-acting factor, as described above.

A virus, such as an adenovirus, also preferably can be a cointegrate,i.e., a ligation of viral, such as adenoviral, genomic sequences withother sequences, such as those of a plasmid, phage or other virus. Interms of an adenoviral vector (particularly a replication-deficientadenoviral vector), such a vector can comprise either complete capsids(i.e., including a viral genome, such as an adenoviral genome) or emptycapsids (i.e., in which a viral genome is lacking, or is degraded, e.g.,by physical or chemical means).

To the extent that it is preferable or desirable to target a virus, suchas an adenovirus, to a particular cell, the virus can be employedessentially as an endosomolytic agent in the transfer into a cell ofplasmid DNA, which contains a marker gene and is complexed and condensedwith polylysine covalently linked to a cell-binding ligand, such astransferrin (Cotten et al., PNAS (USA) 89: 6094-6098 (1992); and Curielet al., PNAS (USA) 88: 8850-8854 (1991)). It has been demonstrated thatcoupling of the transferrin-polylysine/DNA complex and adenovirus (e.g.,by means of an adenovirus-directed antibody, with transglutaminase, orvia a biotin/streptavidin bridge) substantially enhances gene transfer(Wagner et al., PNAS (USA) 89: 6099-6103 (1992)).

Alternatively, one or more viral coat proteins, such as the adenoviralfiber, can be modified, for example, either by incorporation ofsequences for a ligand to a cell-surface receptor or sequences thatallow binding to a bi-specific antibody (i.e., a molecule with one endhaving specificity for the fiber, and the other end having specificityfor a cell-surface receptor) (PCT international patent application no.WO 95/26412 (the '412 application) and Watkins et al., “TargetingAdenovirus-Mediated Gene Delivery with Recombinant Antibodies,” Abst.No. 336). In both cases, the typical fiber/cell-surface receptorinteractions are abrogated, and the virus, such as an adenovirus, isredirected to a new cell-surface receptor by means of its fiber.

Alternatively, a targeting element, which is capable of bindingspecifically to a selected cell type, can be coupled to a first moleculeof a high affinity binding pair and administered to a host cell (PCTinternational patent application no. WO 95/31566). Then, a gene deliveryvehicle coupled to a second molecule of the high affinity binding paircan be administered to the host cell, wherein the second molecule iscapable of specifically binding to the first molecule, such that thegene delivery vehicle is targeted to the selected cell type.

Along the same lines, since methods (e.g., electroporation,transformation, conjugation of triparental mating, (co-)transfection,(co-)infection, membrane fusion, use of microprojectiles, incubationwith calcium phospate-DNA precipitate, direct microinjection; etc.) areavailable for transferring viruses, plasmids, and phages in the form oftheir nucleic acid sequences (i.e., RNA or DNA), a vector similarly cancomprise RNA or DNA, in the absence of any associated protein, such ascapsid protein, and in the absence of any envelope lipid. Similarly,since liposomes effect cell entry by fusing with cell membranes, avector can comprise liposomes, with constitutive nucleic acids encodingthe coat protein. Such liposomes are commercially available, forinstance, from Life Technologies, Bethesda, Md., and can be usedaccording to the recommendation of the manufacturer. Moreover, aliposome can be used to effect gene delivery and liposomes havingincreased transfer capacity and/or reduced toxicity in vivo can be used.The soluble chimeric coat protein (as produced using methods describedherein) can be added to the liposomes either after the liposomes areprepared according to the manufacturer's instructions, or during thepreparation of the liposomes.

In terms of the production of vectors according to the invention(including recombinant adenoviral vectors, recombinant viral vectors andtransfer vectors), standard molecular and genetic techniques, such asthose known to those skilled in the art, are used. Vectors comprisingvirions or viral particles (e.g., recombinant adenoviral vectors) can beproduced using viral vectors in the appropriate cell lines. Similarly,particles comprising one or more chimeric coat proteins can be producedin standard cell lines, e.g., those currently used for adenoviralvectors. These resultant particles then can be targeted to specificcells.

Alterations of the native amino acid sequence to produce variantpeptides can be done by a variety of means known to those skilled in theart. A variant peptide is a peptide that is substantially homologous toa given peptide, but which has an amino acid sequence that differs fromthat peptide. The degree of homology (i.e., percent identity) can bedetermined, for instance, by comparing sequence information using acomputer program optimized for such comparison (e.g., using the GAPcomputer program, version 6.0 or a higher version, described by Devereuxet al. (Nucleic Acids Res. 12: 387 (1984)), and freely available fromthe University of Wisconsin Genetics Computer Group (UWGCG)). Theactivity of the variant proteins and/or peptides can be assessed usingother methods known to those skilled in the art.

In terms of amino acid residues that are not identical between thevariant protein (peptide) and the reference protein (peptide), thevariant proteins (peptides) preferably comprise conservative amino acidsubstitutions, i.e., such that a given amino acid is substituted byanother amino acid of similar size, charge density,hydrophobicity/hydrophilicity, and/or configuration (e.g., Val for Phe).The variant site-specific mutations can be introduced by ligating intoan expression vector a synthesized oligonucleotide comprising themodified site. Alternately, oligonucleotide-directed site-specificmutagenesis procedures can be used, such as those disclosed in Walder etal., Gene 42: 133 (1986); Bauer et al., Gene 37: 73 (1985); Craik,Biotechniques, January 1995: 12-19; and U.S. Pat. Nos. 4,518,584 and4,737,462.

Any appropriate expression vector (e.g., as described in Pouwels et al.,Cloning Vectors: A Laboratory Manual (Elsevior, N.Y.: 1985)) andcorresponding suitable host cell can be employed for production of arecombinant peptide or protein in a host cell. Expression hosts include,but are not limited to, bacterial species within the genera Escherichia,Bacillus, Pseudomonas, Salmonella, mammalian or insect host cellsystems, including baculoviral systems (e.g., as described by Luckow etal., Bio/Technology 6: 47 (1988)), and established cell lines, such asCOS-7, C127, 3T3, CHO, HeLa, BHK, and the like. An especially preferredexpression system for preparing chimeric proteins (peptides) accordingto the invention is the baculoviral expression system whereinTrichoplusia ni, Tn 5B1-4 insect cells, or other appropriate insectcells, are used to produce high levels of recombinant proteins. Theordinary skilled artisan is, of course, aware that the choice ofexpression host has ramifications for the type of peptide produced. Forinstance, the glycosylation of peptides produced in yeast or mammaliancells (e.g., COS-7 cells) will differ from that of peptides produced inbacterial cells, such as Escherichia coli.

Covalently-bound complexes can be prepared by linking a chemical moietyto a functional group on the side chain of an amino acid of a peptide orprotein or at the N- or C-terminus of the peptide or protein. Suchmodifications can be particularly useful, for instance, in constructinga bi-specific or a multi-specific molecule comprising a ligand to acell-surface receptor attached to an antibody. Further modificationswill be apparent to those of ordinary skill in the art.

Viral attachment, entry and gene expression can be evaluated initiallyby using the adenoviral vector containing the insert of interest togenerate a recombinant virus expressing the desired protein or RNA and amarker gene, such as β-galactosidase. β-galactosidase expression incells infected with adenovirus containing the β-galactosidase gene(Ad-LacZ) can be detected as early as two hours after adding Ad-LacZ tocells. This procedure provides a quick and efficient analysis of cellentry of the recombinant virus and gene expression, and is implementedreadily by an artisan of ordinary skill using conventional techniques.

The methods, vectors, modulation systems and compositions of the presentinvention have utility in vitro, such as in the study of viral clearanceand modulation of persistence of transgene expression. Similarly, thepresent inventive methods, vectors, modulation systems and compositionshave utility in vivo. For example, a present inventive vector can beused to treat any one of a number of diseases by delivering to cellscorrective DNA, e.g., DNA encoding a function that is either absent orimpaired. Diseases that are candidates for such treatment include, forexample, cancer, e.g., melanoma or glioma, cystic fibrosis, geneticdisorders, and pathogenic infections, including HIV infection. Otherapplications of the methods and constituents of the present inventionwill be apparent to those skilled in the art.

One skilled in the art will appreciate that many suitable methods ofadministering a vector (i.e., an adenoviral vector or a viral vector),system for modulation or composition of either of the foregoing to ananimal for purposes of gene expression, such as in the context of genetherapy (see, for example, Rosenfeld et al., Science 252: 431-434(1991); Jaffe et al., Clin. Res., 39(2): 302A (1991); Rosenfeld et al.,Clin. Res. 39(2): 311A (1991); Berkner, BioTechniques 6: 616-629 (1988))are available, and, although more than one route can be used foradministration, a particular route can provide a more immediate and moreeffective reaction than another route. Pharmaceutically acceptableexcipients for use in administering a vector also are well-known tothose who are skilled in the art, and are readily available. The choiceof excipient will be determined in part by the particular method used toadminister the vector. Accordingly, the present invention provides acomposition comprising the recombinant at least E4Δ adenoviral vectorand a carrier therefor and a composition comprising the system formodulation of the recombinant at least E4Δ adenoviral vector and acarrier therefor. In this regard, there is a wide variety of suitableformulations for use in the context of the present invention. Thefollowing methods and excipients are merely exemplary and are in no waylimiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachetsor tablets, each containing a predetermined amount of the activeingredient, as solids or granules; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin, emulsions,gels, and the like containing, in addition to the active ingredient,such excipients as are known in the art.

Aerosol formulations can be made for administration via inhalation.These aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. They also can be formulated as pharmaceuticals for non-pressurizedpreparations, such as in a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Additionally, suppositories can be made with the use of a variety ofbases, such as emulsifying bases or water-soluble bases.

Formulations suitable for vaginal administration can be presented aspessaries, tampons, creams, gels, pastes, foams, or spray formulascontaining, in addition to the active ingredient, such carriers as areknown in the art to be appropriate.

The dose administered to an animal, particularly a human, in the contextof the present invention will vary with the transgene of interest, thecomposition employed, the method of administration, and the particularsite and organism being treated. However, preferably, a dosecorresponding to an effective amount of a vector (e.g., an adenoviralvector according to the invention) is employed. An “effective amount” isone that is sufficient to achieve transgene expression in a cell or toproduce a desired effect, e.g., a prophylactic or therapeutic effect, ina host, which can be monitored using several end-points known to thoseskilled in the art. For instance, one desired effect is nucleic acidtransfer to a host cell. Such transfer can be monitored by a variety ofmeans, including, but not limited to, evidence of the transferred geneor coding sequence or its expression within the host (e.g., using thepolymerase chain reaction, Northern or Southern hybridizations, ortranscription assays to detect the nucleic acid in host cells, or usingimmunoblot analysis, antibody-mediated detection, or particularizedassays to detect protein or polypeptide encoded by the transferrednucleic acid, or impacted in level or function due to such transfer) ora therapeutic effect (e.g., alleviation of some symptom associated withthe disease, condition, disorder or syndrome being treated). Thesemethods described are by no means all-inclusive, and further methods tosuit the specific application will be apparent to the ordinary skilledartisan. In this regard, it should be noted that the response of a hostto the introduction of a vector can vary depending on the dose of thevector administered, the site of delivery, and the genetic makeup of thevector as well as the transgene, itself.

Generally, to ensure effective transfer of the vectors of the presentinvention, it is preferred that about 1 to about 5,000 copies of thevector according to the invention be employed per cell to be contacted,based on an approximate number of cells to be contacted in view of thegiven route of administration, and it is even more preferred that about3 to about 300 pfu enter each cell. However, this is merely a generalguideline, which by no means precludes use of a higher or lower amount,as might be warranted in a particular application, either in vitro or invivo. The actual dose and schedule can vary depending on whether thecomposition is administered in combination with other compositions,e.g., pharmaceutical compositions, or depending on interindividualdifferences in pharmacokinetics, drug disposition, and metabolism.Similarly, amounts can vary in in vitro applications depending on theparticular type of cell or the means by which the vector is transferred.One skilled in the art easily can make any necessary adjustments inaccordance with the necessities of the particular situation.

EXAMPLES

The following examples serve to illustrate the present invention and arenot intended to limit its scope in any way.

Example 1

This example demonstrates the level of transgene expression obtained incell culture with an at least E4Δ adenoviral vector comprising secretoryalkaline phosphatase as the transgene and HSV ICP0 or Ad pTP as thetrans-acting factor.

A DNA fragment comprising the coding region of the gene encoding thetrans-acting factor HSV ICP0 or Ad pTP was operably linked to theadenoviral E4 promoter (Ad E4 pro) and an SV40 poly A region in an E4Δadenoviral vector using methods of vector construction known to those ofordinary skill in the art. In addition, the E4Δ adenoviral vector wasmodified to comprise a DNA fragment comprising the coding region of thehuman secretory alkaline phosphatase (SAP) gene operably linked to thecytomegalovirus immediate early promoter (CMVie pro) and an SV40 poly Aregion using well-known methods of vector construction.

Primary human embryonic lung fibroblasts (HEL) cells were infected withone of the following vectors: (i) a mock vector, (ii) an E1ΔE4Δadenoviral vector, (iii) an E1ΔE4Δ adenoviral vector comprising the SAPcoding region operably linked to the CMVie pro and an SV40 poly Aregion, (iv) an E1ΔE4Δ adenoviral vector comprising the SAP codingregion operably linked to the CMVie pro and an SV40 poly A region andthe coding region of HSV ICP0 operably linked to Ad E4 pro and an SV40poly A region, and (v) an E1ΔE4Δ adenoviral vector comprising the SAPcoding region operably linked to the CMVie pro and an SV40 poly A regionand the coding region of Ad pTP operably linked to Ad E4 pro and an SV40poly A region. SAP expression (RLU/2 μl medium) was measured as afunction of multiplicity of infection (moi; particles/cell) to generatea dose-response curve. SAP expression ranged from about 1,000 to about1×10⁷ RLU/2 μl medium for an moi ranging from about 1 to about 1,000particles/cell compared to control (i.e., a mock vector).

Example 2

This example demonstrates that HSV ICP0 can modulate a transgene in anE4Δ adenoviral vector and, thereby, modulate persistence of transgeneexpression in cell culture.

HEL cells were also infected with one of the following vectors: (i) amock vector, (ii) approximately 100 particles/cell of an E1Δ adenoviralvector comprising the SAP coding region operably linked to the CMVie proand an SV40 poly A region, (iii) approximately 1,000 particles/cell of(ii), (iv) approximately 100 particles/cell of an E1ΔE4Δ adenoviralvector comprising the SAP coding region operably linked to the CMVie proand an SV40 poly A region, (v) approximately 1,000 particles/cell of(iv), (vi) approximately 100 particles/cell of an E1ΔE4Δ adenoviralvector comprising the SAP coding region operably linked to the CMVie proand an SV40 poly A region and the coding region of HSV ICP0 operablylinked to AdE4 pro and an SV40 poly A region, and (vii) approximately1,000 particles/cell of (vi) in order to measure SAP expression (RLU/2μl medium) as a function of days post-infection. While expression withSAP was relatively high in HEL cells infected with 1,000 particles/cellof either of the E1Δ adenoviral vector or the E1Δ4Δ adenoviral vector,no expression was detected after around 15 days post-infection. Incontract, SAP expression was maintained at a relatively high level inHEL cells infected with 1,000 particles/cell of the ICP0-expressingE1ΔE4Δ adenoviral vector as long as 28 days post-infection. SAPexpression was also detected at 28 days post-infection in HEL cellsinfected with 100 particles/cell of either of the ICP0-expressing E1ΔE4Δadenoviral vector or the E1Δ adenoviral vector, although atcomparatively lower levels. Substantially lower levels of SAP expressionwas detected in HEL cells infected with 100 particles/cell of E1ΔE4Δadenoviral vector up to about 24 days post-infection. These resultsdemonstrate that HSV ICP0 can modulate a transgene in an E4Δ adenoviralvector and, thereby, modulate persistence of transgene expression.Similar results were also obtained in a human retinal pigmentedepithelial cells (HRPE-19) at approximately 33 days post-infection.

Example 3

This example demonstrates that Ad pTP can modulate a transgene in an E4Δadenoviral vector and, thereby, modulate persistence of transgeneexpression in cell culture.

SAP expression (RLU/2 μl medium) as a function of days post-infectionwas also measured for HEL cells infected with one of the followingvectors: (i) a mock vector, (ii) approximately 100 particles/cell of anE1Δ adenoviral vector comprising the SAP coding region operably linkedto CMVie pro and an SV40 poly A region, (iii) approximately 100particles/cell of an E1ΔE4Δ adenoviral vector comprising the SAP codingregion operably linked to CMVie pro and an SV40 poly A region, (iv)approximately 100 particles/cell of an E1ΔE4Δ adenoviral vectorcomprising the SAP coding region operably linked to CMVie pro and anSV40 poly A region and the coding region of Ad pTP operably linked toAdE4 pro and an SV40 poly A region, and (v) approximately 1,000particles/cell of (iv). While SAP expression was detected at about 28days post-infection for all test vectors except (iii), for whichexpression was not detected past about 24 days post-infection, SAPexpression was highest at 28 days post-infection in cells infected with1,000 particles/cell of Ad pTP-expressing E1ΔE4Δ adenoviral vector.These results demonstrate that Ad pTP can modulate a transgene in an E4Δadenoviral vector and, thereby, modulate persistence of transgeneexpression. Similar results were also obtained in HRPE-19 cells atapproximately 30 days post-infection.

Example 4

This example demonstrates that HSV ICP0 and Ad pTP can modulate atransgene in an intravenously administered E4Δ adenoviral vector and,thereby, modulate persistence of transgene expression in vivo.

Nude mice were intravenously injected with one of the following vectors:(i) approximately 1×10¹⁰ particles of an E1Δ adenoviral vectorcomprising the SAP coding region operably linked to the CMVie pro and anSV40 poly A region, (ii) approximately 1×10¹⁰ particles of an E1ΔE4Δadenoviral vector comprising the SAP coding region operably linked tothe CMVie pro and an SV40 poly A region, (iii) approximately 1×10¹⁰particles of an E1ΔE4Δ adenoviral vector comprising the SAP codingregion operably linked to the CMVie pro and an SV40 poly A region andthe HSV ICP0 coding region operably linked to the E4 promoter and anSV40 poly A region, (iv) approximately 5×10¹⁰ particles of (iii), and(v) approximately 1×10¹⁰ particles of an E1ΔE4Δ adenoviral vectorcomprising the SAP coding region operably linked to the CMVie pro and anSV40 poly A region and the Ad pTP coding region operably linked to theE4 promoter and an SV40 poly A region. At approximately 22 dayspost-infection, high levels of SAP expression was maintained with HSVICP0-expressing and Ad pTP-expressing E4Δ adenoviral vectors.

Example 5

This example demonstrates that HSV ICP0 and Ad pTP can modulate atransgene in an intramuscularly administered E4Δ adenoviral vector and,thereby, modulate persistence of transgene expression in vivo.

Nude mice were intramuscularly injected with one of the followingvectors: (i) approximately 1×10¹⁰ particles of an E1Δ adenoviral vectorcomprising the SAP coding region operably linked to the CMVie pro and anSV40 poly A region, (ii) approximately 5×10¹⁰ particles of (ii), (iii)approximately 1×10¹⁰ particles of an E1ΔE4Δ adenoviral vector comprisingthe SAP coding region operably linked to the CMVie pro and an SV40 polyA region, (iv) approximately 5×10¹⁰ particles of (iii), (v)approximately 1×10¹⁰ particles of an E1ΔE4Δ adenoviral vector comprisingthe SAP coding region operably linked to the CMVie pro and an SV40 polyA region and the HSV ICP0 coding region operably linked to the E4promoter and an SV40 poly A region, (vi) approximately 5×10¹⁰ particlesof (v), and (vii) approximately 1×10¹⁰ particles of an E1ΔE4Δ adenoviralvector comprising the SAP coding region operably linked to the CMVie proand an SV40 poly A region and the Ad pTP coding region operably linkedto the E4 promoter and an SV40 poly A region. At approximately 18 dayspost-infection, high levels of SAP expression was maintained with HSVICP0-expressing and Ad pTP-expressing E4Δ adenoviral vectors.

Example 6

This example demonstrates the ability of a non-Herpes vector comprisinga gene encoding HSV ICP0 to modulate the expression of a transgene in acell in a host by reducing the number of neutralizing antibodiesproduced by the host against the vector.

C57BL/6 mice were given intratracheal administration of either (i)approximately 5×10¹⁰ particles of an E1Δ adenoviral vector comprisingthe SAP coding region operably linked to the CMVie pro and an SV40 polyA region and (ii) approximately 5×10¹⁰ particles of an E1ΔE4Δ adenoviralvector comprising the SAP coding region operably linked to the CMVie proand an SV40 poly A region, and (iii) approximately 5×10¹⁰ particles ofan E1ΔE4Δ adenoviral vector comprising the SAP coding region operablylinked to the CMVie pro and an SV40 poly A region and the HSV ICP0coding region operably linked to the E4 promoter and an SV40 poly Aregion. Neutralizing antibody titer in the serum was measured at 14 daysand 21 days after administration. The approximate averages for eachantibody titer measured at both time points are set forth in Table 1.

TABLE 1 Effect of HSV ICP0 on Neutralizing Antibodies Against AdenoviralVectors (Reciprocal Dilution) Days post E1Δ − E1E4Δ − E1E4Δ −administration HSV ICP0(−) HSV ICP0(−) HSV ICP0(+) 14 days 45 75 15 21days 95 40 30

As can be seen in Table 1, adenoviral vectors comprising a gene encodingHSV ICP0 resulted in a lower production of neutralizing antibodiesproduced against the adenoviral vector by the host than in comparison tosimilar vectors not containing a gene for HSV ICP0. These resultsindicate that non-Herpes vectors, such as adenoviral vectors, comprisinga gene encoding HSV ICP0 result in a lower immune response against theHSV ICP0-expressing vector and thus result in modulation of transgeneexpression.

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein. Theuse of the terms “a” and “an” and “the” and similar referents (e.g.,“the vector” or “an adenoviral vector”) in the context of describing thepresent invention (especially in the context of the following claims)should be construed to cover both the singular and the plural, unlessotherwise indicated herein or clearly contradicted by context.

While this invention has been described with emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat the preferred embodiments may be varied. It is intended that theinvention may be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the appended claims.

What is claimed is:
 1. A method of modulating the persistence ofexpression of a transgene in a cell, wherein the transgene is operablylinked to a Rous sarcoma viral promoter or a cytomegaloviral promoter orthe transgene is part of an expression cassette that comprises such apromoter, which method comprises contacting the cell with a non-Herpesviral vector comprising and expressing a gene encoding HSV ICP0,whereupon expression of HSV ICP0, the persistence of expression of thetransgene is modulated as compared to the persistence of expression ofthe transgene in the absence of expression of HSV ICP0.
 2. The method ofclaim 1, wherein the non-Herpes viral vector further comprises acis-acting element.
 3. The method of claim 1, wherein the transgene ispresent on a viral vector, in which case the method further comprisescontacting the cell with the viral vector.
 4. The method of claim 3,wherein the non-Herpes viral vector and/or the viral vector furthercomprises a cis-acting element.
 5. A system for modulating thepersistence of expression of a transgene, which system comprises: (i) anon-Herpes viral vector comprising a gene encoding HSV ICP0 and (ii) atransgene, wherein the transgene is operably linked to a Rous sarcomaviral promoter or a cytomegaloviral promoter, optionally as part of anexpression cassette, wherein the HSV ICP0 modulates the persistence ofexpression of the transgene and either the non-Herpes viral vectorcomprises the transgene or the transgene is present on a viral vector,in which case the system further comprises the viral vector. 6.(Currently Amended) The system of claim 5, wherein the non-Herpes viralvector and/or the viral vector further comprises a cis-acting element.